RNA interference (RNAi) is a powerful mechanism of post-transcriptional gene silencing originally described in plants and invertebrates as part of an ancient defense for protection against viruses and genetic damage from transposable elements. A little over a year ago RNAi was shown to occur in mammalian cells. It was quickly realized that RNAi might be a useful therapeutic tool to treat viral infection. Several laboratories, including the principal investigators in this program, showed that RNAi could suppress replication of HIV and other RNA viruses in vitro. Our group also showed that small interfering RNAs (siRNAs) targeting fas could be delivered in vivo to protect mice from fulminant hepatitis in autoimmune models. Although this in vivo study used chemically synthesized dsRNAs, more recently it has been possible to express siRNAs from stem loop structures encoded by plasmids, retroviruses and lentiviruses. These vectors provide a means for in vivo transduction of a variety of cells and expand the possible therapeutic uses for RNAi. Most NIAID priority viral pathogens are RNA viruses, which should be ideal targets for RNAi. This program will study both fundamental and applied aspects of RNAi, since many of its fundamental features in mammalian cells are still unknown or poorly understood. The overall goal of this multidisciplinary, integrated program is to determine whether RNAi can be used to combat bioterrorism. In particular, the program aims to understand the basic mechanisms of RNAi and its interaction with viral infection, to determine the therapeutic approaches, and anticipate some of the problems that may arise. The program will lay the groundwork for the subsequent evaluation of RNAi in nonhuman primates and in human pilot proof-of-principle clinical studies. The four projects probe the mechanisms for generation and function of siRNA and its close relative, microRNA (miRNA), understanding the effect of viral infection on RNAi and its interaction with the interferon response, developing inducible and tissue-specific vector-based delivery systems and studying in vivo applicability of siRNA-based strategies to target host and viral genes. The program will examine both universal strategies to prevent death acutely by inhibiting immunoregulatory genes that contribute in a stereotypic way to septic shock and fulminant hepatitis from many pathogens, as well as targeting specific viral bioterrorism agents, such as flaviviruses and poxviruses. A mouse biohazard core will provide facility and expertise to handle Bioterrorism Category A-C agents in an already existing BL3 tissue culture and animal facility, which will be expanded, reequipped and renovated to handle the anticipated increased use.